Project Summary Botulinum neurotoxins (BoNTs) are among the most deadly bacterial toxins, which cause the neuroparalytic disease botulism. They are categorized as Tier 1 select agents by the CDC, and could be misused as biological weapons. An antitoxin composed of equine-derived polyclonal IgG antibodies is currently the only available treatment for botulism. Since the equine antitoxin can cause various side effects, substantial efforts have been invested to develop monoclonal human antibody antitoxins. These antibodies are effective at neutralizing circulating BoNTs thereby preventing further disease progression. But antibodies are ineffective for BoNTs that have become internalized into neurons by the time botulism symptom is observed in patients (generally 24?48 hours following exposure). Despite extensive research, no small-molecule or peptidomimetic inhibitors for BoNTs has been advanced to clinical trials. Therefore, there is an urgent need for novel approaches to the prevention and treatment of BoNT intoxication. In this proposal, we will focus on the type A toxin (BoNT/A) that poses the most serious threat to humans because of its high potency and long duration of action. The goal is to develop the first high-throughput, large-scale computational design and screening of small cyclic peptides, which inhibit the protease activity of the light chain of BoNT/A (LC/A) with high affinity and specificity. These cyclic peptides combine the specificity of antibodies with the high stability and manufacturability of small molecules, and could be optimized to improve membrane permeability to enter the nerve termini. Furthermore, they are highly resistant to proteases and high environmental temperature, thus could bypass the requirement for cold chain management. The specific aims are (1) to perform large-scale de novo design of hyperstable peptide inhibitors targeting LC/A; and (2) to characterize the peptide inhibitors identified in Aim 1 and elucidate the co-crystal structures of LC/A with the best binders. This project will be carried out using an integrated approach that combines Rosseta design, micro-chip gene synthesis, multiplex yeast cell surface display screening platform, next-generation deep sequencing, X-ray crystallography, and in vitro binding and protease assays.